Bacillus anthracis, the causative agent of anthrax, is a spore-forming, Gram-positive, non-hemolytic, rod-shaped bacterium. Anthrax is primarily a zoonotic disease of herbivores; however, humans can naturally acquire this disease directly from contact with infected herbivores, or indirectly via their products, such as hair, wool, and hides. Spores are the usual infective form. Anthrax presents clinically as three distinct syndromes, depending on the route of infection: cutaneous, gastrointestinal, and inhalational disease. Cutaneous anthrax is the most common naturally occurring form in humans. However, inhalational anthrax, although seen only rarely in naturally acquired infections, would be the major concern in a situation involving the release of aerosolized spores. Such was demonstrated by the accidental release of aerosolized spores from Sverdlovsk in the former Soviet Union in 1979 (Meselson et al 1994) and the intentional release of aerosolized spores in the anthrax letter attacks in the United States in October 2001 (Jernigan et al., 2001). The high level of mortality seen with inhalational anthrax can be mitigated by administration of the appropriate antibiotics within 24-48 hours of exposure. However, delays in administrating antibiotics beyond 24-48 hours of exposure generally results in death to individuals receiving a lethal dose of spores.
The spore coat and exosporium of B. anthracis have been the focus of previous studies. When B. anthracis vegetative cells are deprived of essential nutrients (“starved”), a trigger is given to begin synthesis of the endospore (“spore”). The following sequence of events takes place when the vegetative cells are starved: 1) an asymmetric septation of starved vegetative cells occurs, resulting in the formation of the mother cell and a forespore; 2) the mother cell engulfs the forespore, thus surrounding the forespore with two opposing cell membranes; 3) a thick layer of modified peptidoglycan (“cortex”) is synthesized between the two membranes; and 4) proteins synthesized in the mother cell form multiple layers of a spore coat that covers the cortex.
The spore coat forms the outermost layer for spores of some Bacillus species, such as B. subtilis. However, in other species, such as B. anthracis, the spore is enclosed by an additional layer called the exosporium, a loose balloon-like layer containing proteins, lipid, and carbohydrate. Charlton et al. (“Characterization of the exosporium of Bacillus cereus” J. App. Microbiol. 87:241-245, 1999) describe studies on the exosporium of B. cereus. Spores of the closely related species B. thuringiensis also have an exosporium. A number of investigators have previously identified spore coat and exosporium antigens of B. anthracis. Lai et al. (“Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis” J. Bact., (185(4):1443-1454, 2003), using proteomic analysis employing a combination of SDS-PAGE separation and 2-D 313ctrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF®), identified 38 spore proteins of B. subtilis (of which 12 are known spore coat proteins) and 11 spore proteins of B. anthracis (6 of which they identified as candidate coat or exosporium proteins). From their studies comparing B. subtilis and B. anthracis spore proteins, Lai et al. concluded that “B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species” (underlining added; see Lai et al. abstract).
Steichen et al. (“Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium”, J. Bact., 185(6):1903-1910, 2003) identified five major proteins in purified B. anthracis exosporium, including the collagen-like-glycoprotein BclA, which they described as a structural component of the exosporium hair-like nap. These investigators concluded that BclA is the immunodominant antigen on the B. anthracis spore surface because 12 out of 20 monoclonal antibodies raised against either spores or purified exosporium reacted with BclA. The other four proteins identified by Steichen et al. are alanine racemase, superoxide dismutase, and two proteins with no significant similarity to any other protein, which they called BxpA and BxpB.
In addition, Todd et al. (“Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium”, J. Bact., 185(11):3373-3378, 2003) evaluated exosporium proteins of B. cereus. B. cereus is a member of the Bacillus cereus family, which includes B. thuringiensis and B. anthracis, all of which possess an exosporium and all of which are close relatives. Other related Bacillus species include B. subtilis, B. globigii, B. pumilis, B. mycoides, and B. megaterium. Todd et al. identified 10 exosporium proteins of B. cereus. They concluded, based on a comparative analysis of B. cereus protein sequences with predicted protein sequences from the B. anthracis genome sequences that “[f]rom the available unfinished genome sequences, most of the novel Exs proteins are closely conserved between B. cereus and B. anthracis, with two exceptions . . . a local region of ExsB and the entire ExsC protein that may not be expressed in B. anthracis.” (see page 3378, first full paragraph). They further note that their “identified genes do not by any means represent an exhaustive list of protein components of the exosporium; one-third of protein remained in the insoluble fraction, and 7 out of 17 bands have not yielded clear N-terminal sequence data.” (see page 3378, fourth full paragraph).
In the literature concerning spore coat or exosporium proteins of B. anthracis, the only monoclonal antibodies developed were to the immunodominant Bacillus collagen-like protein of anthracis, BclA (see Sylvestre et al., “A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium” Molec. Microbiol. 45(1):169-178, 2002; and Steichen et al.). The spore specific monoclonal antibody of the instant invention as described herein does not react with this protein. Longchamp et al. (“Molecular recognition specificity of Bacillus anthracis spore antibodies” J. App. Microbiol. 87:246-249, 1999) describe the characterization of polyclonal serum which recognized a wide range of spore surface epitopes which cross-reacted with related Bacillus species. They further describe two monoclonal antibodies that did not react with spore surface epitopes. Lee et al. (WO 01/49823) describe antibodies against a B. anthracis surface array protein, to which the 23a-14G9 monoclonal antibody of the instant invention as described below does not react.
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